Antisense Oligonucleotides
The first generation of antisense oligonucleotides were intended to affect the activity of target mRNAs. One reason for interest in such oligonucleotides is the potential for exquisite and predictable specificity that can be achieved because of specific base pairing. It is in theory very simple to design an oligonucleotide that is highly specific for a given nucleic acid, such as an mRNA. However simple base pairing is not enough to achieve regulation of a given target mRNA. That is, an oligonucleotide complementary to a given target mRNA does not necessarily affect the activity of the target mRNA. If the oligonucleotide targets the open reading frame of an mRNA, it may, for example, be that the translational apparatus simply displaces the oligonucleotide during translation. Therefore, means were developed that would improve the regulatory activity of the oligonucleotide, including oligonucleotides that can activate RNase H cleavage of the target mRNA. However one disadvantage of such oligonucleotides is that they may mediate cleavage of RNAs other than the intended target mRNA giving rise to off-target effects. Notwithstanding, several oligonucleotides acting through RNase H cleavage are in clinical trial for the treatment of various diseases.
More recently research has shown that eukaryotic cells, including mammalian cells, comprise a complex gene regulatory system (RNAi machinery) that uses RNA as specificity determinants. This system can be triggered by small interfering RNA (siRNA) that may be introduced into a cell of interest to regulate the activity of a target mRNA. Currently, considerable effort goes into using siRNA as novel therapeutics to trigger RNAi machinery for specific regulation of target RNAs, in particular target mRNAs. However siRNAs are proving less specific than initially thought and result in significant off-target effects. It is now believed that these off-targets stem from the siRNAs, or rather the guide strand of the siRNAs, acting as microRNAs.
MicroRNAs
MicroRNAs (miRNAs) are a class of endogenous RNA molecules that, like siRNA, function via the RNAi machinery. miRNAs are small, single stranded, non-coding RNAs which regulate both mRNA degradation and translation, at least partially through their ability to bind to the 3′UTR of target genes through base pairing with the 5′-end of the miRNA, via the so called seed sequence or seed region of the miRNA.
Currently, more than 500 human miRNAs have been discovered and it is believed that more than one third of all human genes are regulated by miRNAs. Therefore, miRNAs themselves may be used to regulate the activity of target RNAs, opening the opportunity to develop miRNAs as therapeutics. However, miRNAs generally act at more than one target RNA; they are promiscuous. Thus, introduction of a miRNA into a cell or regulating the level of a miRNA in a cell will affect the activity of more than one target mRNA and consequently may give rise to unexpected and undesired effects.
miRNAs can be inhibited using complementary oligonucleotides, termed antimirs and antagomirs. However since each miRNA is itself promiscuous, any given antimir or antagomir will similarly affect the activity of more than one target mRNA.
miRNA and Angiogenesis
The tight control of vascular permeability is one of the chief functions of the endothelial lining of blood vessels. A loss of this barrier function of endothelium underlies many general and organ specific disease processes including leakiness of tumor vessels, various respiratory distress syndromes, complications of chemotherapy as well as acute anaphylactoid reactions. Controls of permeability generally fall into two classes: one the stimuli, like thrombin, histamine, vascular endothelial growth factor (VEGF), that induce leakiness. The other, a class of molecules like angiopoetin-1 that exert a tonic effect on sustaining the status quo. Ultimately these two influences converge on the cell-cell junctional molecules such as VE Cadherin and PECAM that regulate junctional structure.
Given the relatively clear phenotypes induced by these stimuli it is surprising how self-limited the leakiness is under circumstances of repair or physiological angiogenesis. The inventors reasoned that there may exist yet undiscovered mechanisms that will operate to force a quick restoration to normality of vessels, for example in the process of angiogenesis, or that operate during physiological angiogenesis to limit the leakiness of newly forming vessels. This would be in stark contrast to tumour angiogenic vessels that are characterized by excessive leakiness, where these control mechanisms may be by-passed.
In the vasculature, miRNAs have been linked to regulation of development, and to diseases such as tumour growth and cardiovascular disease. For example, the endothelial cell-restricted miRNA, miR-126, is highly expressed during vascular development and tumour angiogenesis, while in contrast miR-101 is downregulated in tumour vessels, acting through histone-methyltransferase to promote angiogenesis. In endothelial cells miR-296 is VEGF responsive and blockade of this miRNA inhibits tumour-associated angiogenesis. miR-132 is induced in tumour angiogenic vessels, targeting p120RasGAP which acts downstream of integrins to increase cell proliferation and vascular growth. miR-92a targets integrin signalling and its inhibition improves blood flow recovery following ischemic insult.